Bromodomain inhibition decreases IL7R expression and suppresses JAK2/STAT5 phosphorylation. (A) Immunoblotting of whole cell lysates from cells treated with 500nM JQ1 at indicated time points. (B) Quantitative PCR of CRLF2 transcript levels in MHH-CALL4 and MUTZ-5 cells treated with 500nM JQ1. Data for each time point are normalized to vehicle control and presented as a ratio of expression compared with baseline expression at time = 0. Error bars represent ± SEM. *P < .1 (paired t test). **P < .01 (paired t test). (C) Quantitative PCR analysis of IL7R transcripts levels, as in panel B. (D) Flow cytometry for CRLF2 and IL7R. Cells were treated with 500nM JQ1 or vehicle for 8 and 24 hours and stained with an antibody against the indicated target or an isotype IgG. (E) ChIP with a BRD4 antibody at 2 sites within the IL7R promoter region in cells treated with 500nM JQ1 for 4 hours. Enrichment is shown as the percentage of total input DNA. Negative control region primers amplify within a gene desert region ∼ 70 kb downstream of IL7R. *P < .1 (unpaired t test). **P < .01 (unpaired t test). (F) Quantitative PCR of MYC and IL7R expression and (G) flow cytometry of IL7R expression in B-ALL cell lines that lack CRLF2 rearrangements after 8-hour treatment with 500nM JQ1 or vehicle, as above.